The present invention relates to a composition and method to elicit an immune response in a mammal using a genetic immunization strategy. More particularly, the present invention includes compositions and methods for eliciting systemic, non-specific (i.e., non-antigen-specific) immune responses in a mammal as well as antigen-specific immune responses, both of which are useful in immunization protocols.
Vaccines are widely used to prevent disease and to treat established diseases (therapeutic vaccines). There remains, however, an urgent need to develop safe and effective vaccines and adjuvants for a variety of diseases, including those due to infection by pathogenic agents, cancers and other disorders amenable to treatment by elicitation of an immune response.
Three major types of disease in mammals which are amenable to elicitation and/or modulation of an immune response include infectious diseases, allergic inflammatory diseases and cancer, although the present invention is not limited to treatment of these disease types. Infectious diseases are caused by infectious agents (i.e., infectious disease pathogens), examples of which include viruses, bacteria, parasites, yeast and other fungi. In allergic inflammatory diseases, allergens cause the release of inflammatory mediators that recruit cells involved in inflammation in allergic or sensitized animals, the presence of which can lead to tissue damage and sometimes death. Cancer can result from an inherited inability to repair DNA, to prevent DNA damage or to prevent propagation of cells with damaged DNA, and/or from a biochemical dysfunction or genetic mutation which leads to uncontrolled cell proliferation and DNA synthesis.
Traditional reagents that are used in an attempt to protect a mammal from such diseases include reagents that destroy infectious agents or the cells involved in deregulated biological functions, or that modify the activity of such cells. Such reagents, however, can result in unwanted side effects. For example, anti-viral drugs that disrupt the replication of viral DNA also often disrupt DNA replication in normal cells in the treated patient. The use of anti-inflammatory and symptomatic relief reagents in allergic inflammation is a serious problem because of their side effects or their failure to attack the underlying cause of an inflammatory response. Other treatments with chemotherapeutic reagents to destroy cancer cells typically leads to side effects, such as bleeding, vomiting, diarrhea, ulcers, hair loss and increased susceptibility to secondary cancers and infections.
An alternative method of disease treatment includes modulating the immune system of a patient to assist the patient""s natural defense mechanisms. Traditional reagents and methods used to attempt to regulate an immune response in a patient also result in unwanted side effects and have limited effectiveness. For example, immunopharmacological reagents used to treat cancer (e.g., interleukins) are short-lived in the circulation of a patient and are ineffective except in large doses. Due to the medical importance of immune regulation and the inadequacies of existing immunopharmacological reagents, reagents and methods to regulate specific parts of the immune system have been the subject of study for many years.
Vaccines can be used not only to prevent disease, but can also be used to treat established diseases (i.e., therapeutic vaccines). A number of tumor antigens which are recognized by T lymphocytes of the immune system have been recently identified and are being considered as potential vaccine candidates. Conventional vaccines generally consist of either (1) purified antigens administered with an adjuvant, or (2) an attenuated form of a pathogen that can be administered to a patient to generate an immune response, but not cause serious disease or illness.
Genetic vaccines, by contrast, contain a DNA sequence that encodes an antigen(s) against which the immune response is to be generated. For genetic vaccines to generate an antigen-specific immune response, the gene of interest must be expressed in the mammalian host. Gene expression has been accomplished by use of viral vectors (e.g., adenovirus, poxvirus) that express the foreign gene of interest in the vaccinated patient and induce an immune response against the encoded protein. Alternatively, plasmid DNA encoding a foreign gene has been used to induce an immune response. The primary routes of administration of these so-called xe2x80x9cnakedxe2x80x9d DNA vaccines are intramuscular or percutaneous. It is generally accepted that viral vector systems induce better immune responses than naked DNA systems, probably because the viral delivery systems induce more inflammation and immune activation than naked DNA vaccines. The propensity of viral vaccines to induce non-specific immune responses, primarily as a result of viral component recognition by the complement cascade, also represents a potential drawback, however, since such immune responses often prevent readministration of the vaccine.
Therefore, there is need to provide better vaccines which can produce an immune response which is safe, antigen-specific and effective to prevent and/or treat diseases amenable to treatment by elicitation of an immune response, such as infectious disease, allergy and cancer.
One embodiment of the present invention generally relates to a method to elicit a systemic, non-antigen-specific immune response in a mammal. The method includes the step of administering to the mammal a therapeutic composition by a route of administration selected from intravenous and intraperitoneal administration. The therapeutic composition includes: (a) a liposome delivery vehicle; and, (b) an isolated nucleic acid molecule that is not operatively linked to a transcription control sequence. In another embodiment, the route of administration is intravenous. In further embodiments of the method, the isolated nucleic acid molecule comprises a non-coding sequence. In one embodiment, the isolated nucleic acid molecule does not comprise a bacterial nucleic acid sequence.
Accordingly, another embodiment of the present invention is a composition for eliciting a systemic, non-antigen-specific immune response in a mammal. Such a composition includes (a) a liposome delivery vehicle; and (b) an isolated nucleic acid molecule that is not operatively linked to a transcription control sequence. In one embodiment, the nucleic acid molecule does not include a bacterial nucleic acid sequence.
Another embodiment of the present invention relates to a composition for eliciting a systemic, non-antigen-specific immune response in a mammal which comprises (a) a liposome delivery vehicle and (b) an isolated non-coding nucleic acid sequence.
A composition of the present invention can further comprise a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient can include, for example a non-ionic diluent, and more preferably, 5 percent dextrose in water (D5W).
The above-mentioned method and compositions of the present invention have the advantages of eliciting a systemic, non-antigen specific immune response in a mammal, and more particularly, of eliciting a systemic, anti-viral immune response in a mammal. Additionally, the method and composition of the present invention can elicit a systemic, anti-tumor immune response in a mammal. Such an anti-tumor immune response can result in the reduction of a tumor in the mammal. The method and composition of the present invention can also elicit a systemic, protective immune response against allergic inflammation in a mammal. The systemic, non-antigen-specific immune response elicited by the method and composition of the present invention result in an increase in effector cell activity, and particularly, natural killer (NK) cell activity in the mammal, and additionally can result in increased production of IFNxcex3 in the mammal.
Yet another embodiment of the present invention relates to a method to elicit an immunogen-specific immune response and a systemic, non-specific immune response in a mammal. The method includes administering to the mammal a therapeutic composition by a route of administration selected from intravenous and intraperitoneal. The therapeutic composition comprises: (a) a liposome delivery vehicle; and, (b) a recombinant nucleic acid molecule comprising an isolated nucleic acid sequence encoding an immunogen, wherein the nucleic acid sequence is operatively linked to a transcription control sequence. Particularly suitable transcription control sequences include Rous sarcoma virus (RSV) control sequences, cytomegalovirus (CMV) control sequences, adenovirus control sequences and Simian virus (SV-40) control sequences. This method of the present invention has the particular advantage of eliciting both a systemic, non-immunogen-specific immune response in a mammal, as well as an immunogen-specific immune response that have a potent therapeutic effect in the mammal. In one embodiment, the route of administration is intravenous. In other preferred embodiments, the immunogen is a tumor antigen, an infectious disease pathogen antigen or an allergen.
When the mammal has cancer, this immunogen is preferably a tumor antigen. In one embodiment of this method, the therapeutic composition can include a plurality of recombinant nucleic acid molecules, each of the recombinant nucleic acid molecules comprising a cDNA sequence amplified from total RNA isolated from an autologous tumor sample, each of the cDNA sequences encoding a tumor antigen or a fragment thereof and being operatively linked to a transcription control sequence. In another embodiment, the therapeutic composition comprises a plurality of recombinant nucleic acid molecules, each of the recombinant nucleic acid molecules comprising a cDNA sequence amplified from total RNA isolated from a plurality of allogeneic tumor samples of the same histological tumor type, each of the cDNA sequences encoding a tumor antigen or a fragment thereof and being operatively linked to a transcription control sequence.
The methods and compositions of the present invention are particularly useful for treating a cancer which includes melanomas, squamous cell carcinoma, breast cancers, head and neck carcinomas, thyroid carcinomas, soft tissue sarcomas, bone sarcomas, testicular cancers, prostatic cancers, ovarian cancers, bladder cancers, skin cancers, brain cancers, angiosarcomas, hemangiosarcomas, mast cell tumors, primary hepatic cancers, lung cancers, pancreatic cancers, gastrointestinal cancers, renal cell carcinomas, hematopoietic neoplasias, and metastatic cancers thereof. The compositions and methods of the present invention are especially useful for treating primary lung cancer or pulmonary metastatic cancer.
Accordingly, a tumor antigen useful in the present composition is preferably from a cancer selected from the group of melanomas, squamous cell carcinoma, breast cancers, head and neck carcinomas, thyroid carcinomas, soft tissue sarcomas, bone sarcomas, testicular cancers, prostatic cancers, ovarian cancers, bladder cancers, skin cancers, brain cancers, angiosarcomas, hemangiosarcomas, mast cell tumors, primary hepatic cancers, lung cancers, pancreatic cancers, gastrointestinal cancers, renal cell carcinomas, hematopoietic neoplasias and metastatic cancers thereof. The tumor antigen preferably is selected from the group of tumor antigens having epitopes that are recognized by T cells, tumor antigens having epitopes that are recognized by B cells, tumor antigens that are exclusively expressed by tumor cells, and/or tumor antigens that are expressed by tumor cells and by non-tumor cells.
When the immunogen is a tumor antigen which is expressed in the mammal, the method of the present invention produces a result selected from alleviation of the cancer, reduction of size of a tumor associated with the cancer, elimination of a tumor associated with the cancer, prevention of metastatic cancer, prevention of the cancer and stimulation of effector cell immunity against the cancer. When the tumor antigen is administered intravenously, the antigen is expressed in a pulmonary tissue of the mammal and prevents pulmonary metastatic cancer in the mammal.
When the immunogen is an infectious disease pathogen antigen, the methods and composition of the present invention are useful for mammals having an infectious disease, and particularly for mammals having a chronic infectious disease. Such immunogens can be from infectious disease pathogens which include bacteria, viruses, parasites and fungi. Such infectious disease pathogens include, for example, human immunodeficiency virus (HIV), Mycobacterium tuberculosis, herpesvirus, papillomavirus and Candida. The present method is particularly useful when the infectious disease pathogen is a virus, and more particularly, human immunodeficiency virus and feline immunodeficiency virus. In another embodiment, the present method is particularly useful when the infectious disease is tuberculosis. In this embodiment, the immunogen can be, for example, a Mycobacterium tuberculosis antigen, or more specifically, antigen 85.
Expression of the pathogen antigen in a tissue of the mammal produces a result selected from the group of alleviation of the disease, regression of established lesions associated with the disease, alleviation of symptoms of the disease, immunization against the disease and/or stimulation of effector cell immunity against the disease.
In one embodiment of this method, the therapeutic composition comprises a plurality of recombinant nucleic acid molecules, each of the recombinant nucleic acid molecules comprising a cDNA sequence amplified from total RNA isolated from an infectious disease pathogen, each of the cDNA sequences encoding an immunogen from the infectious disease pathogen or a fragment thereof and being operatively linked to a transcription control sequence.
When the mammal has a disease associated with allergic inflammation, the immunogen is an allergen. Suitable allergens include, plant pollens, drugs, foods, venoms, insect excretions, molds, animal fluids, animal hair and animal dander. This method is particularly useful when the mammal has a disease selected from allergic airway diseases, allergic rhinitis, allergic conjunctivitis, and food allergy. Expression of the allergen in a tissue of the mammal produces a result selected from the group consisting of alleviation of the disease, alleviation of symptoms of the disease, desensitization against the disease, and stimulation of a protective immune response against the disease.
In another embodiment of this method, the therapeutic composition comprises a plurality of recombinant nucleic acid molecules, each of the recombinant nucleic acid molecules comprising a cDNA sequence amplified from total RNA isolated from an allergen, each of the cDNA sequences encoding the allergen or a fragment thereof and being operatively linked to a transcription control sequence.
Yet another embodiment of the present invention relates to a method to elicit a systemic, non-specific immune response in a mammal, which includes administering to the mammal a therapeutic composition by a route of administration selected from intravenous and intraperitoneal, wherein the therapeutic composition comprises: (a) a liposome delivery vehicle; and, (b) a recombinant nucleic acid molecule comprising an isolated nucleic acid sequence encoding a cytokine, the nucleic acid sequence being operatively linked to a transcription control sequence. The method of the present invention is particularly useful for eliciting a systemic, anti-viral immune response or a systemic; an anti-tumor immune response; a systemic, protective immune response against allergic inflammation in the mammal; and/or for reduction of a tumor in the mammal. Additionally, the method increases production of IFNxcex3 in the mammal and/or increases natural killer (NK) cell activity in the mammal. In one embodiment, the route of administration is intravenous. The cytokine can include hematopoietic growth factors, interleukins, interferons, immunoglobulin superfamily molecules, tumor necrosis factor family molecules and/or chemokines. In one embodiment, the cytokine is an interleukin, and in a more preferred embodiment, the interleukin is selected from the group of interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18) or interferon-xcex3 (IFNxcex3), and in an even more preferred embodiment, the interleukin is selected from the group of interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-18 (IL-18) or interferon-xcex3 (IFNxcex3).
Another embodiment of the present invention relates to a method to elicit a tumor antigen-specific immune response and a systemic, non-specific immune response in a mammal that has cancer. The method includes administering to a mammal a therapeutic composition by a route of administration selected from intravenous and intraperitoneal administration. The therapeutic composition comprises: (a) a liposome delivery vehicle; and, (b) total RNA isolated from a tumor sample, the RNA encoding tumor antigens. In one embodiment, the route of administration is intravenous. In another embodiment, the RNA is enriched for poly-A RNA prior to administration to the mammal.
Yet another embodiment of the present invention relates to a method to elicit a pathogen-antigen-specific immune response and a systemic, non-specific immune response in a mammal that has an infectious disease. Such method includes administering to a mammal a therapeutic composition by a route of administration selected from intravenous and intraperitoneal administration, the therapeutic composition comprising: (a) a liposome delivery vehicle; and, (b) total RNA isolated from an infectious disease pathogen, the RNA encoding pathogen antigens. In another embodiment, the route of administration is intravenous.
Another embodiment of the present invention relates to a composition for systemic administration to a mammal to elicit an immunogen-specific immune response and a systemic, non-specific immune response. The composition includes (a) a liposome delivery vehicle; and (b) a recombinant nucleic acid molecule comprising an isolated nucleic acid sequence encoding an immunogen, the nucleic acid sequence being operatively linked to a transcription control sequence. The composition has a nucleic acid:lipid ratio of from about 1:1 to about 1:64.
In one embodiment, any of the above compositions of the present invention administered to a mammal by the present methods can include a recombinant nucleic acid molecule having a nucleic acid sequence encoding a cytokine. In this embodiment, the nucleic acid sequence encoding a cytokine is operatively linked to a transcription control sequence. In the compositions which include a nucleic acid sequence encoding an immunogen, the nucleic acid sequence encoding a cytokine can be in the same or separate recombinant nucleic acid molecule which contains the nucleic acid sequence encoding the immunogen. The nucleic acid sequence-encoding a cytokine and the nucleic acid sequence encoding an immunogen can be operatively linked to the same or different transcription control sequences. In preferred embodiments, the cytokine is selected from the group of hematopoietic growth factors, interleukins, interferons, immunoglobulin superfamily molecules, tumor necrosis factor family molecules and/or chemokines. In one embodiment, the cytokine is an interleukin, and in a more preferred embodiment, the interleukin is selected from the group of interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18) or interferon-xcex3 (IFNxcex3), and in an even more preferred embodiment, the interleukin is selected from the group of interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-18 (IL-18) or interferon-xcex3 (IFNxcex3).
Liposome delivery vehicles suitable for use in any of the compositions and methods of the present invention can include any liposomes. Particularly preferred liposomes are cationic liposomes. Other preferred liposomes include multilamellar vesicle lipids and extruded lipids, with multilamellar vesicle lipids being more preferred. Liposome compositions can include, but are not limited to, pairs of lipids selected from DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol, with DOTAP and cholesterol being particularly preferred.
The compositions of the present invention administered by the present methods have a nucleic acid:lipid ratio of from about 1:1 to about 1:64. In some embodiments, the compositions have a nucleic acid:lipid ratio of from about 1:10 to about 1:40. Other suitable ratios are additionally set forth below.
The methods and compositions of the present invention are preferably used to elicit an immune response in a mammal, which includes humans, dogs, cats, mice, rats, sheep, cattle, horses or pigs, and more preferably, humans.